María Esther Escudero

Effectiveness of various disinfectants in the elimination of Yersinia enterocolitica on fresh lettuce

The effectiveness of various disinfectants against two potentially pathogenic Yersinia enterocolitica strains (Y. enterocolitica W1024 O:9 [strain A] and Y. enterocolitica B O:5 Lis Xz [strain B]) on shredded lettuce was examined. Dip-wash treatments using 25, 100, and 300 ppm of chlorine at 4 and 22 degrees C, 0.2% Orenco Peel 40, 0.1% Tergitol, 0.5% acetic acid, and 0.5% lactic acid at 22 degrees C were performed. Surfactants and organic acids were also tested in combination with 100 ppm of chlorine. Reductions of Y. enterocolitica counts with 100 ppm (2.68 log10 for strain A and 2.36 log10 for strain B at 22 degrees C) and 300 ppm of chlorine (3.15 log10 for strain A and 2.55 log10 for strain B at 4 degrees C) were observed after 10 min. Inhibitory effect of different chlorine solutions was not significantly (P < 0.05) influenced by temperature. Surfactants in combination with chlorine were more effective than surfactants alone. Treatment with 0.2% Orenco Peel 40 plus 100 ppm of chlorine resulted in reductions of 2.69 log10 CFU/g for strain A and 3.18 1og10 CFU/g for strain B at 10 min. Dip solutions containing 0.1% Tergitol plus 100 ppm of chlorine produced a significant reduction of 2.73 log10 CFU/g in strain A (P < 0.05). With the 0.5% lactic acid plus 100 ppm of chlorine combination, inactivation of Y. enterocolitica was >6 log10. The bactericidal effect of disinfectants was related to the concentration, exposure time, combination with chlorine (surfactants and organic acids), and susceptibility of each strain. Since the presence of pathogenic Y. enterocolitica on ready-to-use vegetables represents a health hazard, treatments as effective as 0.5% lactic acid plus 100 ppm of chlorine are recommended for washing of fresh lettuce.

Genotypic and phenotypic characteristics of Yersinia enterocolitica isolated from the surface of chicken eggshells obtained in Argentina.

The prevalence of Yersinia enterocolitica on chicken eggshell surfaces in San Luis, Argentina, was investigated. The pathogenic potential of recovered isolates was assessed by means of phenotypic virulence tests and the presence of the 72-kb pYV plasmid. Antimicrobial susceptibility was determined by the agar diffusion method. DNA digested with XbaI was analyzed by pulsed-field gel electrophoresis (PFGE), and relationships between genomic DNA profiles were established. Eight Y. enterocolitica B2 O:9 strains were recovered after enrichment, for a prevalence of 2.27%. All strains harbored the virulence pYV plasmid, bound Congo red, grew in a low-calcium medium, and autoagglutinated at 37 degrees C. They lacked pyrazinamidase activity and did not hydrolyze esculin. These Y. enterocolitica strains were susceptible to amikacin, ciprofloxacin, chloramphenicol, and trimethoprim-sulfamethoxazole and were resistant to rifampin. According to the genomic DNA patterns obtained by PFGE, the isolates clustered into two groups, I and II. The highest similarity coefficient observed between Y. enterocolitica strains was 0.947. Microbiological controls on production stages of eggs and good culinary practices are necessary to reduce the risk of Y. enterocolitica infection for consumers.

Effect of Chlorine, Sodium Chloride, Trisodium Phosphate, and Ultraviolet Radiation on the Reduction of Yersinia enterocolitica and Mesophilic Aerobic Bacteria from Eggshell Surface

Eggshell sanitizing practices are necessary to improve microbiological safety of fresh hen eggs and their products. In this work, the effects of 100 mg/liter free chlorine (chl), 3% sodium chloride (NaCl), 1, 5, and 12% trisodium phosphate (TSP) in wash solutions, and UVR (ultraviolet radiation; 4.573 microW/cm2) were studied at different times on uninoculated and Yersinia enterocolitica-inoculated eggs. On uninoculated eggs, the best results were obtained with 100 mg/liter chlorine and UV exposure for >25 min, with reductions of 1.28 and 1.60 log cycles, respectively, compared to the average bacterial count (4.55 log CFU/egg) on the control (untreated eggs). On Y. enterocolitica-inoculated eggs, highest reductions of the average bacterial count (7.35 log CFU/egg) were obtained with 5 and 12% TSP and 100 mg/liter chl. The decrease obtained with 12% TSP (3.74-log reduction) was significantly higher (P < 0.05) than those obtained with the remaining treatments. Y. enterocolitica was more resistant to UVR than the eggshell natural mesophilic aerobic microflora, except when low inoculum (4.39 log CFU/egg) was assayed. Changes in eggshell microstructure were measured by the blue lake staining method. The presence of Yersinia and Salmonella in eggshell natural flora was also investigated.

Survival of Yersinia enterocolitica and Mesophilic Aerobic Bacteria on Eggshell after Washing with Hypochlorite and Organic Acid Solutions

Populations of Yersinia enterocolitica 0:9 and mesophilic aerobic bacteria on the shell of fresh chicken eggs were assessed prior and after washing with 0.75%, 1%, and 3% acetic and lactic acids, 50, 100, and 200 mg/liter (ppm) of chlorine, and water. Highest reductions of mesophilic aerobic bacterial populations (normal flora) on trypticase soy agar were 1.28 and 2.15 log10 cycles with 100 and 200 mg/liter of chlorine, 0.28 and 0.36 log10 cycles with 1% and 3% acetic acid, and 0.70 and 0.71 log10 cycles with 1% and 3% lactic acid, respectively, as compared to the control group. No Salmonella or Yersinia were detected among the natural flora of the eggs. On Y. enterocolitica O:9-inoculated eggs, reductions of 2.66, 2.77, and 2.92 log10 cycles by 50, 100, and 200 mg/liter of chlorine, of 2.47, 2.48, and 2.49 log10 cycles by 0.75%, 1%, and 3% of acetic acid, and of 2.48 and 2.72 log10 cycles with 1% and 3% of lactic acid, respectively, were observed with respect to the control. Organic acids at 3% caused detachment of the surface cuticle of the eggshell. Y. enterocolitica was more sensitive to the wash treatments than the natural microflora. The absence of potentially pathogenic Y. enterocolitica, observed for other fresh foods, should be a norm for fresh eggs sold in retail stores.

Pulsed field, PCR ribotyping and multiplex PCR analysis of Yersinia enterocolitica strains isolated from meat food in San Luis Argentina

The characterization of phenotypic and genotypic virulence markers of Yersinia enterocolitica strains belonging to biotypes (B) 1A, 2 and 3, mostly isolated from food in San Luis, Argentina, and the assessment of their genotypic diversity using PFGE and PCR ribotyping, were performed in our laboratory for the first time. Thirty five Y. enterocolitica strains, two reference strains and 33 strains isolated in our laboratory were studied. The presence of virF, ail, ystA, and myfA genes was investigated by multiplex PCR. The pathogenic potential of B1A strains, the most predominant biotype of Y. enterocolitica strains isolated from meat in our region, was investigated by simple PCR. Four B1A strains were positive for ystB gene. Four Y. enterocolitica 2/O:9 (bio/serotype) and two 3/O:5 strains isolated in our laboratory showed virulence-related results in the phenotypic tests and multiplex PCR. A good correlation between the expression of virulence markers and their corresponding genotypes was observed for most strains. Sixteen genomic types (GT) and 9 different intergenic spacer region (SR) groups were generated by PFGE and PCR ribotyping, respectively. In both cases the Y. enterocolitica 2/O:9 strains were separately clustered from 1A and 3/O:5 strains. Meat foods might be vehicles of transmission of pathogenic Y. enterocolitica strains in our region.

Prevalence of Yersinia enterocolitica in hake (Merluccius hubbsi) fillets

Yersinia enterocolitica is a human pathogenic bacterium. The prevalence of Y. enterocolitica in refrigerated hake fillets sold for human consumption in retail stores was investigated in order to determine the degree of pathogenicity. Three hundred samples were enriched in 0.067 M phosphate-buffered saline, pH 7.6, with 1% sorbitol and 0.15% biliary salts, postenriched in 0.5% KOH, and isolated in salmonella-shigella agar and MacConkey agar. Twelve strains of Yersinia were isolated from the whole group of samples, 11 (91.6%) of which corresponded to Y. enterocolitica and 1 (8.3%) to Y. intermedia . The Yersinia strains recovered were Y. enterocolitica B1 O:5 Lis Xz (1 strain), Y. enterocolitica B3 O:5 (1 strain), Y. enterocolitica B1 O:6,30-6,31 (1 strain), Y. enterocolitica B1 O:7,8-8-8,19 (1 strain), Y. enterocolitica B1 O:7,8-8-8,19 Lis Xz (7 strains) and Y. intermedia B1 O:40 Lis Xo (1 strain). Of the 12 strains isolated, 8 (66.6%) were recovered by alkaline postenrichment. The first two strains were positive for virulence tests (autoagglutination and calcium dependence for growth at 37°C). The antibiotic susceptibility of the isolated strains was studied by the agar-diffusion method according to Bauer-Kirby, modified by Barry. Some of the isolated strains were potentially pathogenic, representing a risk for human health.

Antibacterial Effects of Different Food-Related Phosphates Using Aeromonas hydrophila

Aeromonas hydrophila is considered to be an emergent food-related bacterium. Phosphates are used as additives, mainly in meat products, to improve the quality of these foods. The antibacterial properties of phosphates are also well known. In this work, two A. hydrophila strains in early exponential phase were used: (A) A. hydrophila ATCC 7965 and (B) A. hydrophila derived from food, isolated in our laboratory. MIC and MBC studies were performed to assess the antibacterial effects of four phosphates assayed in brain heart infusion broth (BHI) and modified complete defined synthetic medium (mCDS) as compared to cooked ground meat medium (CM). The MBC values of the phosphates in CM were significantly higher than MIC values in BHI broth and mCDS medium (P < 0.05). In the two latter media, the growth of both A. hydrophila strains was totally inhibited by concentrations between 0.5 and 3.0%. Although all the assayed phosphates proved to have bactericidal effects on A. hydrophila, 0.5% sodium acid pyrophosphate (SAPP) exhibited greater effects in both strains and was selected for subsequent experiments. The bacteriolytic effect of SAPP was spectrophotometrically determined (260 nm of absorbance) by means of the leakage of intracellular nucleotides and microscopically confirmed by the presence of massive gelatinous aggregates. These were identified by enzymes (RNase, DNase, and proteinase) that hydrolyzed the nucleotides and proteins released during cellular lysis in the presence of SAPP. It was concluded that 0.5% SAPP can have bactericidal and bacteriolytic effects in early exponential phase A. hydrophila cells.

Survival of Aeromonas hydrophila in fresh tomatoes (Lycopersicum esculentum Mill) stored at different temperatures and treated with chlorine.

This study examines the survival of two Aeromonas hydrophila strains (A. hydrophila ATCC 7965 [strain A] and A. hydrophila isolated from food [strain B] on the surface and core tissue of fresh tomatoes stored at different temperatures and the efficacy of chlorine treatment on their survival. Counts of A. hydrophila on the surface of tomatoes stored at 25 and 35 degrees C were significantly higher between days 1 and 4 for both strains as compared to results obtained at 6 degrees C. Core tissue counts of A. hydrophila cells on tomatoes dipped in a cellular suspension at 25 degrees C and stored at 25 degrees C were significantly higher (P < 0.05) than counts obtained with dip suspensions at 6 or 35 degrees C. In chopped tomatoes stored at 25 and 35 degrees C, populations of aerobic mesophiles showed significant increases after 96 and 70 h, respectively. The populations of both A. hydrophila strains in chopped tomatoes stored at 6 degrees C increased significantly after 96 h, while at 25 and 35 degrees C the counts increased in the first hours of incubation. Viable counts of A. hydrophila on the surface and central tissue of tomatoes significantly decreased (P < 0.05) when the samples were dipped for 2 min in chlorine at a concentration of 50 ppm (50 microgram/ml). The results suggest that tomatoes should be kept at low temperatures during storage, shipping, and retail stocking and that chlorine at a concentration of 50 ppm should be used to reduce the levels of A. hydrophila.

Intranasal Immunization with Yersinia enterocolitica O:8 Cellular Extract Protects against Local Challenge Infection

Yersinia enterocolitica is enteropathogenic for humans and rodents. Immune protection from oral and respiratory pathogens may be most effectively elicited following intranasal (i.n.) vaccination. An experimental murine intranasal challenge model was used to evaluate the immunogenicity of a Y. enterocolitica O:8 cellular extract (CE) in mucosa. This antigenic preparation has demonstrated to induce protection by subcutaneous immunization. Mice were immunized intranasally with two doses of CE. Immunized and nonimmunized animals were challenged with 5×106 colony‐forming units (CFU) by nasal infection. Antibodies in serum and bronchoalveolar lavage (b.a.l.) fluid were assessed before and 48 hr after challenge. The CFU were determined by analysis of lung homogenate samples. The CE immunization induced significant b.a.l.‐specific IgA and IgG, and serum‐specific IgG, IgA and IgM. Histopathological studies 24 and 48 hr postchallenge demonstrated that immunization protected against progressive lesions resulting from Y. enterocolitica invasion of the pulmonary mucosa. The CFU in the lungs showed that CE immunization led to significant clearance as compared to the bacterial level in nonimmunized controls. From the results obtained, it can be concluded that CE can induce local and systemic immunity and protect against nasal infection.

Detection and Characterization of Shiga Toxin Producing Escherichia coli, Salmonella spp., and Yersinia Strains from Human, Animal, and Food Samples in San Luis, Argentina

Shiga toxin producing Escherichia coli (STEC), Salmonella spp., and Yersinia species was investigated in humans, animals, and foods in San Luis, Argentina. A total of 453 samples were analyzed by culture and PCR. The antimicrobial susceptibility of all the strains was studied, the genomic relationships among isolates of the same species were determined by PFGE, and the potencial virulence of Y. enterocolitica strains was analyzed. Yersinia species showed higher prevalence (9/453, 2.0%, 95% CI, 0.7–3.3%) than STEC (4/453, 0.9%, 95% CI, 0–1.8%) and Salmonella spp. (3/453, 0.7%, 95% CI, 0–1.5%). Y. enterocolitica and Y. intermedia were isolated from chicken carcasses (6/80, 7.5%, 95% CI, 1.5–13.5%) and porcine skin and bones (3/10, 30%, 95% CI, 0–65%). One STEC strain was recovered from human feces (1/70, 1.4%, 95% CI, 0–4.2%) and STEC stx1/stx2 genes were detected in bovine stools (3/129, 2.3%, 95% CI, 0–5.0%). S. Typhimurium was isolated from human feces (1/70, 1.4%, 95% CI, 0–4.2%) while one S. Newport and two S. Gaminara strains were recovered from one wild boar (1/3, 33%, 95% CI, 0–99%). The knowledge of prevalence and characteristics of these enteropathogens in our region would allow public health services to take adequate preventive measures.